新型转基因减毒疟疾疫苗的构建与抗疟机理研究
|
摘要
疟疾是一种古老的寄生虫传染病,严重威胁着人类健康,阻碍着社会发展。据WHO最新公布数字,全球有一半人口受到疟疾的威胁,2008年有2.43亿人感染疟原虫,约86.3万人因患疟疾死亡,大多是5岁以下的儿童。近年来,虽然在疟疾控制方面取得了显著的成绩,但由于疟原虫抗药性以及蚊媒对杀虫剂抗性的产生和扩散,使疟疾防治面临严重困难。当前普遍认为,研制安全、价廉和有效的疫苗是人类控制乃至根除疟疾的重要途径。世界卫生组织、联合国计划开发署、世界银行等已将疟疾疫苗研究项目作为全球优先发展的三大疫苗项目之一。
疟疾感染从雌性按蚊叮咬人体,其唾液腺中的成熟疟原虫子孢子随蚊虫的唾液注入宿主血液循环开始,随后子孢子进入肝细胞,在其中行裂体增殖而形成裂殖体。成熟后肝细胞破裂,裂殖子被释放入血液,进入红细胞并在其中行裂体增殖。经过若干增殖周期后致使红细胞破裂,出现临床症状。由此可见,子孢子是疟疾感染人类宿主的上游环节,以子孢子作为疟疾疫苗引起有效的免疫应答可阻断子孢子进入肝细胞从而终止疟疾生活史,达到有效防治疟疾的目的。
疟疾疫苗的开发研究已有40多年的历史,经历了全虫疫苗(包括死疫苗和放射减毒活疫苗)、基因工程亚单位疫苗和化学合成的多肽疫苗、核酸及病原载体疫苗等几个时期。但是,所有这些疫苗在人体试验中都没有达到理想的效果,其中疫苗的安全性、诱导的免疫应答特异性和效价不高、免疫力持续时间短等都是其中原因之一。如何兼顾疫苗的安全性及增高疟疾疫苗的免疫反应能力,延长疫苗免疫反应的时间是疟疾研究疫苗研究的重要内容。
Mueller等人用基因敲除的方法将疟原虫UIS3基因敲除后,发现缺失了UIS3基因的子孢子具有减毒功能,感染动物模型后疟原虫子孢子停留于肝细胞期而不发展为红细胞期疟疾而终止疟疾生活史,将其作为减毒全虫疫苗,在小鼠疟疾模型中可以诱导产生十分理想的免疫保护作用。本研究利用基因打靶技术敲除伯氏疟原虫的UIS3基因,同时为了获得良好的免疫效果,利用转基因技术将具有免疫增强作用的B淋巴细胞趋化因子(B-lymphocytechemoattractant,BLC)转进基因工程减毒的伯氏疟原虫UIS3基因敲除质粒中再转染疟原虫子孢子,以期获得一种既有减毒,又能够增加免疫功能的新型疟原虫株(UIS3-/BLC+)。
BLC是近年新发现的一种特异性趋化B细胞的细胞因子,并能刺激细胞表达趋化因子受体BLR1,参与引导B细胞的游走和归巢,能吸引B细胞和CD4+T细胞趋化到免疫应答部位从而增强特异免疫应答。将BLC基因修饰疟疾疫苗,则可增强针对疟原虫的特异性体液免疫应答及细胞免疫应答。
本研究根据基因库提供的小鼠BLC基因序列设计克隆扩增BLC的cDNA基因序列的引物,利用RT-PCR技术获得小鼠BLC cDNA基因,通过限制性内切酶酶切和T4酶连接,将BLC基因cDNA序列插入伯氏疟原虫UIS3基因敲除质粒中,获得重组伯氏疟原虫BLC转基因重组质粒。重组质粒含有息疟定抗性基因和绿色荧光蛋白的报告基因,可在转化子中表达。筛选重组质粒并经酶切和DNA序列测定证实正确性后,体外转染真核细胞。经琼脂糖凝胶电泳发现扩增的小鼠BLC基因cDNA序列分子量大小和预期值相一致,重组质粒酶切结果和预期值相符,DNA序列测定显示重组质粒的目的基因阅读框架准确无误。从mRNA蛋白质表达二个层面证明重组质粒可以在真核细胞中有效表达BLC基因。证明已成功构建了伯氏疟原虫BLC转基因重组质粒(UIS3-/BLC+)。
伯氏疟原虫在转化前经体外短期培养,利用密度梯度介质Renografin分离子孢子用于电穿孔转化。将构建的伯氏疟原虫BLC转基因重组质粒经酶切线性化后转染伯氏疟原虫,获得UIS3-/BLC+疟原虫株。将此疟原虫株经尾静脉注入小鼠体内,用息疟定腹腔内注射进行药物筛选,经2轮筛选后,得到了阳性的伯氏疟原虫转化子。在荧光显微镜下观察到经息疟定筛选的原虫呈现绿色荧光；经PCR检测到了重组质粒的存在,说明重组质粒已正确的整合在伯氏疟原虫基因之中。
本研究进一步在小鼠伯氏疟疾模型中,采用分子生物学、免疫学和组织学等技术对获得的UIS3-/BLC+疟原虫株进行抗疟机理研究。结果发现UIS3-/BLC+疟原虫株可以在肝细胞期有效生长,但未能有效发育转化为红内期,具备了基因敲除减毒的特性。与UIS3子孢子免疫相比,我们发现,UIS3-/BLC+子孢子免疫的小鼠可以有效产生体液和细胞免疫反应,抗体的水平和持续时间均明显强于UIS3-子孢子,T淋巴细胞特异杀伤疟原虫的能力也明显增强。体内外抗疟疾感染试验结果也表明,UIS3-/BLC+子孢子作为疫苗较UIS3-具有更好的抑制疟原虫生长和转化的作用。
本研究结果表明,基因工程敲除并转入免疫增强基因的UIS3-/BLC+疟原虫子孢子是一种很好的疟疾疫苗模式,具有较好的临床应用前景,值得深入研究。
Malaria is a parasitic disease with long history, posing a serious threat to human health and social development. According to the data offered by World Heath Organization (WHO),243 million cases of Plasmodium infection were reported and about 863 thousand patients died of malaria in 2008, of which the majorities were younger than 5 years old. It has achieved greatly in the control of malaria. However, the diagnosis and the treatment are still difficult due to drug-resistance of Plasmodium and the resistance of vectors to pesticide. Generally, the safe, cheap and effective vaccine is considered as the key strategy in controlling and eliminating malaria. So WHO, United Nations Development Programe, World Bank etc. have listed the development of vaccine against malaria as one of three prior vaccine projects.
Malaria infection starts from the biting of the female anopheles, by which sporozoites of Plasmodium with saliva are injected into the hosts'blood circulation. Then the sporozoites enter hepatic cells and are turned into schizonts by schizogony. Mature schizonts cause the rupture of hepatic cells and are released into the blood. These schizonts experience another schizogony in erythrocyte. They cause erythrocyte fragmentation after several multiplication cycles, and then lead to the occurrence of clinical symptoms, such as periodically fever and anemia. So sporozoite is an important upstream stat in infection process. The effective immunity of sporozoite vaccine can block the entering of sporozoite into hepatic cells, terminate the development of further infection, prevent and treat the malaria effectively.
Vaccines against malaria have been developed for more than 40 years, and the research experienced whole Plasmodium vaccine period (killed vaccine and live radiation-attenuated vaccine), genetic engineering subunit vaccine and chemosynthesis polypeptide vaccine period, nucleic acid and pathogen vector vaccine period etc. However, tests show that these vaccines are not safe or effective as expected due to low specificity of immunological response, low titer specific antibodies, short duration of immunity, etc. Thus, increasing the safety and strengthening the immunological ability and prolonging the reaction time are essential for vaccine research.
Mueller and their colleagues found that sporozoites of Plasmodium with UIS3 gene knockout were attenuated. After infecting animal models, these sporozoites stayed in hepatocells not in erythrocytes. They can be used as attenuated vaccine, and have immuno-protective effect on mice with malaria. In this study, we knocked UIS3 gene out in Plasmodium berghei (P.b) by gene targeting technique. In order to gain a good immunity, we transferred B-lymphocyte chemoattractant(BLC) gene into the attenuated P.b plasmid, and then transferred the new plasmid into sporozoites by transgenic technique. Thus, we obtained a new strain, UIS3-/BLC+, which is attenuated and can strengthen immunological ability.
BLC is a specific chemokine of B cells. It can stimulate B-lymphocyte receptor-1 expression, guide the migration and homing of B cells, promote immune response by attracting B cells and CD4+T cells. Modification by BLC gene can enhance both specific humoral immunity and cellullar immunologic response to Plasmodium.
According to BLC sequence of mice from genetic library, the primer was designed. BLC cDNA was amplified by PR-PCR. Then the amplified cDNA was inserted into the plasmid of Plasmodium with UIS3 knockout linked by restriction endonuclease and T4 ligase. This recombinant plasmid contains Pyrimethamine-resistant gene and report gene of green fluorescent protein, and can be expressed in transformant. After screening, digestion and sequencing, the recombinant plasmid was transfected into eukaryocyte in vitro. Analysis of agarose gel electrophoresis showed that the molecular weight of cDNA and the digestion result of recombinant plasmid were the same as expected. DNA sequencing also confirmed the open-reading frame of target gene. It proved from both mRNA and protein levels that BLC gene can be expressed in eukaryocyte.
Before transformation, Plasmodium was cultured in vitro for short term. While during mature schizont period, schizont was separated by a density gradient mediator, Nycoprep, for transformation by electroporation. After digesting recombinant plasmid and transfecting into Plasmodium, a new strain was screened (pUIS3-/BLC+). After transfection by recombinant plasmid, Plasmodium was injected into mice via caudal vein, and Pyrimethamine was also injected intraperitoneally for screening. The positive transformant was obtained after twice screening, and displayed green fluorescence under fluorescence microscope. PCR test also revealed recombinant plasmid, confirming the integration of plasmid and Plasmodium gene.
Selecting Plasmodium with pUIS3-/BLC+ as the vaccine, and mice infected by Plasmodium as the model, we focused on the anti-malaria mechanism by molecular biological, immunological and histological methods in this study. The results show that Plasmodium with pUIS3-/BLC+ can grow in hepatocyte, but it can't develop into erythrocytic stage. So it is attenuated with gene knockout. Comparing with immune reaction induced by sporozoite with UIS3-, mice with pUIS3-/BLC+ can produce effective humoral immune reaction and cellular immune reaction, with significantly higher titer and longer duration of antibody, and stronger T cell respones. Anti-malaria test in vivo and in vitro all prove that sporozoite with pUIS3-/BLC+ is more capable to depress the growth and transformation of Plasmodium.
This study shows that sporozoite of Plasmodium with gene knockout and transferred immunoenhancing gene provides good model for development of vaccine against malaria. It is promising in clinical application and is worthy further research.
疟疾感染从雌性按蚊叮咬人体,其唾液腺中的成熟疟原虫子孢子随蚊虫的唾液注入宿主血液循环开始,随后子孢子进入肝细胞,在其中行裂体增殖而形成裂殖体。成熟后肝细胞破裂,裂殖子被释放入血液,进入红细胞并在其中行裂体增殖。经过若干增殖周期后致使红细胞破裂,出现临床症状。由此可见,子孢子是疟疾感染人类宿主的上游环节,以子孢子作为疟疾疫苗引起有效的免疫应答可阻断子孢子进入肝细胞从而终止疟疾生活史,达到有效防治疟疾的目的。
疟疾疫苗的开发研究已有40多年的历史,经历了全虫疫苗(包括死疫苗和放射减毒活疫苗)、基因工程亚单位疫苗和化学合成的多肽疫苗、核酸及病原载体疫苗等几个时期。但是,所有这些疫苗在人体试验中都没有达到理想的效果,其中疫苗的安全性、诱导的免疫应答特异性和效价不高、免疫力持续时间短等都是其中原因之一。如何兼顾疫苗的安全性及增高疟疾疫苗的免疫反应能力,延长疫苗免疫反应的时间是疟疾研究疫苗研究的重要内容。
Mueller等人用基因敲除的方法将疟原虫UIS3基因敲除后,发现缺失了UIS3基因的子孢子具有减毒功能,感染动物模型后疟原虫子孢子停留于肝细胞期而不发展为红细胞期疟疾而终止疟疾生活史,将其作为减毒全虫疫苗,在小鼠疟疾模型中可以诱导产生十分理想的免疫保护作用。本研究利用基因打靶技术敲除伯氏疟原虫的UIS3基因,同时为了获得良好的免疫效果,利用转基因技术将具有免疫增强作用的B淋巴细胞趋化因子(B-lymphocytechemoattractant,BLC)转进基因工程减毒的伯氏疟原虫UIS3基因敲除质粒中再转染疟原虫子孢子,以期获得一种既有减毒,又能够增加免疫功能的新型疟原虫株(UIS3-/BLC+)。
BLC是近年新发现的一种特异性趋化B细胞的细胞因子,并能刺激细胞表达趋化因子受体BLR1,参与引导B细胞的游走和归巢,能吸引B细胞和CD4+T细胞趋化到免疫应答部位从而增强特异免疫应答。将BLC基因修饰疟疾疫苗,则可增强针对疟原虫的特异性体液免疫应答及细胞免疫应答。
本研究根据基因库提供的小鼠BLC基因序列设计克隆扩增BLC的cDNA基因序列的引物,利用RT-PCR技术获得小鼠BLC cDNA基因,通过限制性内切酶酶切和T4酶连接,将BLC基因cDNA序列插入伯氏疟原虫UIS3基因敲除质粒中,获得重组伯氏疟原虫BLC转基因重组质粒。重组质粒含有息疟定抗性基因和绿色荧光蛋白的报告基因,可在转化子中表达。筛选重组质粒并经酶切和DNA序列测定证实正确性后,体外转染真核细胞。经琼脂糖凝胶电泳发现扩增的小鼠BLC基因cDNA序列分子量大小和预期值相一致,重组质粒酶切结果和预期值相符,DNA序列测定显示重组质粒的目的基因阅读框架准确无误。从mRNA蛋白质表达二个层面证明重组质粒可以在真核细胞中有效表达BLC基因。证明已成功构建了伯氏疟原虫BLC转基因重组质粒(UIS3-/BLC+)。
伯氏疟原虫在转化前经体外短期培养,利用密度梯度介质Renografin分离子孢子用于电穿孔转化。将构建的伯氏疟原虫BLC转基因重组质粒经酶切线性化后转染伯氏疟原虫,获得UIS3-/BLC+疟原虫株。将此疟原虫株经尾静脉注入小鼠体内,用息疟定腹腔内注射进行药物筛选,经2轮筛选后,得到了阳性的伯氏疟原虫转化子。在荧光显微镜下观察到经息疟定筛选的原虫呈现绿色荧光；经PCR检测到了重组质粒的存在,说明重组质粒已正确的整合在伯氏疟原虫基因之中。
本研究进一步在小鼠伯氏疟疾模型中,采用分子生物学、免疫学和组织学等技术对获得的UIS3-/BLC+疟原虫株进行抗疟机理研究。结果发现UIS3-/BLC+疟原虫株可以在肝细胞期有效生长,但未能有效发育转化为红内期,具备了基因敲除减毒的特性。与UIS3子孢子免疫相比,我们发现,UIS3-/BLC+子孢子免疫的小鼠可以有效产生体液和细胞免疫反应,抗体的水平和持续时间均明显强于UIS3-子孢子,T淋巴细胞特异杀伤疟原虫的能力也明显增强。体内外抗疟疾感染试验结果也表明,UIS3-/BLC+子孢子作为疫苗较UIS3-具有更好的抑制疟原虫生长和转化的作用。
本研究结果表明,基因工程敲除并转入免疫增强基因的UIS3-/BLC+疟原虫子孢子是一种很好的疟疾疫苗模式,具有较好的临床应用前景,值得深入研究。
Malaria is a parasitic disease with long history, posing a serious threat to human health and social development. According to the data offered by World Heath Organization (WHO),243 million cases of Plasmodium infection were reported and about 863 thousand patients died of malaria in 2008, of which the majorities were younger than 5 years old. It has achieved greatly in the control of malaria. However, the diagnosis and the treatment are still difficult due to drug-resistance of Plasmodium and the resistance of vectors to pesticide. Generally, the safe, cheap and effective vaccine is considered as the key strategy in controlling and eliminating malaria. So WHO, United Nations Development Programe, World Bank etc. have listed the development of vaccine against malaria as one of three prior vaccine projects.
Malaria infection starts from the biting of the female anopheles, by which sporozoites of Plasmodium with saliva are injected into the hosts'blood circulation. Then the sporozoites enter hepatic cells and are turned into schizonts by schizogony. Mature schizonts cause the rupture of hepatic cells and are released into the blood. These schizonts experience another schizogony in erythrocyte. They cause erythrocyte fragmentation after several multiplication cycles, and then lead to the occurrence of clinical symptoms, such as periodically fever and anemia. So sporozoite is an important upstream stat in infection process. The effective immunity of sporozoite vaccine can block the entering of sporozoite into hepatic cells, terminate the development of further infection, prevent and treat the malaria effectively.
Vaccines against malaria have been developed for more than 40 years, and the research experienced whole Plasmodium vaccine period (killed vaccine and live radiation-attenuated vaccine), genetic engineering subunit vaccine and chemosynthesis polypeptide vaccine period, nucleic acid and pathogen vector vaccine period etc. However, tests show that these vaccines are not safe or effective as expected due to low specificity of immunological response, low titer specific antibodies, short duration of immunity, etc. Thus, increasing the safety and strengthening the immunological ability and prolonging the reaction time are essential for vaccine research.
Mueller and their colleagues found that sporozoites of Plasmodium with UIS3 gene knockout were attenuated. After infecting animal models, these sporozoites stayed in hepatocells not in erythrocytes. They can be used as attenuated vaccine, and have immuno-protective effect on mice with malaria. In this study, we knocked UIS3 gene out in Plasmodium berghei (P.b) by gene targeting technique. In order to gain a good immunity, we transferred B-lymphocyte chemoattractant(BLC) gene into the attenuated P.b plasmid, and then transferred the new plasmid into sporozoites by transgenic technique. Thus, we obtained a new strain, UIS3-/BLC+, which is attenuated and can strengthen immunological ability.
BLC is a specific chemokine of B cells. It can stimulate B-lymphocyte receptor-1 expression, guide the migration and homing of B cells, promote immune response by attracting B cells and CD4+T cells. Modification by BLC gene can enhance both specific humoral immunity and cellullar immunologic response to Plasmodium.
According to BLC sequence of mice from genetic library, the primer was designed. BLC cDNA was amplified by PR-PCR. Then the amplified cDNA was inserted into the plasmid of Plasmodium with UIS3 knockout linked by restriction endonuclease and T4 ligase. This recombinant plasmid contains Pyrimethamine-resistant gene and report gene of green fluorescent protein, and can be expressed in transformant. After screening, digestion and sequencing, the recombinant plasmid was transfected into eukaryocyte in vitro. Analysis of agarose gel electrophoresis showed that the molecular weight of cDNA and the digestion result of recombinant plasmid were the same as expected. DNA sequencing also confirmed the open-reading frame of target gene. It proved from both mRNA and protein levels that BLC gene can be expressed in eukaryocyte.
Before transformation, Plasmodium was cultured in vitro for short term. While during mature schizont period, schizont was separated by a density gradient mediator, Nycoprep, for transformation by electroporation. After digesting recombinant plasmid and transfecting into Plasmodium, a new strain was screened (pUIS3-/BLC+). After transfection by recombinant plasmid, Plasmodium was injected into mice via caudal vein, and Pyrimethamine was also injected intraperitoneally for screening. The positive transformant was obtained after twice screening, and displayed green fluorescence under fluorescence microscope. PCR test also revealed recombinant plasmid, confirming the integration of plasmid and Plasmodium gene.
Selecting Plasmodium with pUIS3-/BLC+ as the vaccine, and mice infected by Plasmodium as the model, we focused on the anti-malaria mechanism by molecular biological, immunological and histological methods in this study. The results show that Plasmodium with pUIS3-/BLC+ can grow in hepatocyte, but it can't develop into erythrocytic stage. So it is attenuated with gene knockout. Comparing with immune reaction induced by sporozoite with UIS3-, mice with pUIS3-/BLC+ can produce effective humoral immune reaction and cellular immune reaction, with significantly higher titer and longer duration of antibody, and stronger T cell respones. Anti-malaria test in vivo and in vitro all prove that sporozoite with pUIS3-/BLC+ is more capable to depress the growth and transformation of Plasmodium.
This study shows that sporozoite of Plasmodium with gene knockout and transferred immunoenhancing gene provides good model for development of vaccine against malaria. It is promising in clinical application and is worthy further research.
引文
1. Snow RW, Guerra CA, Noor AM, et al. The global distribution of clinical episodes of plasmodium falciparum malaria [J]. Nature,2005; 434 (7030):214-217.
2. WHO World Malaria Report 2009[EB/OL].http://www.who.int/malaria/world_malaria_report_2009/en/index.html.
3. Kappe SH, Kaiser K, Matuschewski K. The Plasmodium sporozoite journey: a rite of passage [J]. Trends Parasitol,2003; 19:135-143.
4.李雍龙主编,人体寄生虫学(第七版)[M].北京：人民卫生出版社,2008.
5.周家莲,杨恒林.抗疟药研究现状与发展趋势[J].中国病原生物学杂志,2008,11(3)：865-7.
6. Marsh K, Kinyanjui S. Immune effector mechanisms in malaria [J]. Parasite Immunol,2006,28(1/2):51-60.
7.周华,孙立新,朱昌亮.疟疾疫苗研究的主要进展[J].国外医学寄生虫病分册,2005,32(2)：72-6.
8. Danie J, Carucci MC. Malaria vaccine research at the Naval Medical ResearchCenter.Navy Med,2001,5：23.
9.潘卫庆.疟疾疫苗研究的现状和展望[J].第二军医大学学报,2004,25(1)：1-4.
10. Nussenzweig RS,Vanderberg J,Most H,et al. Protective immunity produced by the injection of X-irradiated sporozoites of Plasmodium berghei [J]. Nature, 1967; 216:160-162.
11.朱艳琴,叶炳辉.疟疾疫苗的研究现状和难度[J].微生物学免疫学进展,1997,25(4)：74-6.
12. Thathy V., Menard R..Gene Targeting in Plasmodium berghei. Methods Mol Med,2002;72:317-331.
13. Girard MP, Reed ZH, FriedeM, KienyMP. A review of human vaccine research and development:malaria. Vaccine 2007; 25:1567-1580.
14. Renia L, Gruner AC, Mauduit M, Snounou G. Vaccination against malaria with live parasites. Expert Rev Vaccines 2006; 5:473-481.
15. SedegahM, Hoffman SL. Immunological responses of neonates and infants to DNA vaccines. Methods Mol Med 2006; 127:239-251.
16. Matuschewski K. Vaccine development against malaria. Curr Opin Immunol 2006; 18:449-457.
17. Holling Dale M R. Antisporozoite antibodies[J]. Bull WHO,1990,68 (5):47-48.
18. Hoffman SL,Goh LM,Luke TC,et al.Protection of humans against malaria by immunization with radiation-attenuated Plasmodium falciparum sporozoites [J]. J Infect Dis,2002;185:1155-64.
19. Glyde D F. Immunity to Plasmodium falciparum an vivax malaria induced by irra-diated sporozoites:are view of the university o f Maryland studies[J]. Bull WHO,1990,68 (5):9-12.
20. Srivastava K, Singh S, Singh P, Puri SK. In vitro cultivation of Plasmodium falciparum:studies with modified medium supplemented with ALBUMAX II and various animal sera. Exp Parasitol 2006; [Epub ahead of print].
21. Balu B, Adams JH. Advancements in transfection technologies for plasmodium. Int J Parasitol 2007; 37:1-10.
22. Scheller LF,Azad AF.Maintenance of protective immunity against malaria by persistent hepatic parasites derived from irradiated sporozoites [J]. Proc Natl Acad Sci USA,1995;92:4066-4068.
23. Luke TC, Hoffman SL. Rationale and plans for developing a nonreplicating, metabolically active, radiation-attenuated Plasmodium falciparum sporozoite vaccine. J Exp Biol 2003; 206:3803-3808.
24. Mueller AK, Labaied M, Kappe SH, et al. Genetically modified Plasmodium parasites as a protective experimental malaria vaccine [J]. Nature,2005; 433 (7022):164-167.
25. Mueller AK,Camargo N,Kaiser K,et al.Plasmodium liver stage developmental arrest by depletion of a protein at the parasite-host interface[J].Proc Natl Acad Sci USA,2005,102(8):3022-3027.
26. Van Dijk MR, Douradinha B, Franke-Fayard B,et al. Genetically attenuated, P36p-deficient malarial sporozoites induce protective immunity and apoptosis of infected liver cells[J].Proc Natl Acad Sci USA,2005,102(34):12194-12199.
27. Daubersies P,Thomas AW,Millet P, et al.Protection against Plasmodium falciparum malaria in chimpanzees by immunization with the conserved pre-erythrocytic liver-stage antigen 3 [J]. Nat Med,2000;6:1258-63.
28. Alonso PL,Sacarlal J,Aponte JJ,et al.Efficacy of the RTS,S/AS02A vaccine against Plasmodium falciparum infection and disease in young African children: randomized controlled trial [J]. Lancet,2004;364:1411-1420.
29. Enea V, Ellis J, Zavala F, et al. DNA cloning of Plasmodium falciparum circumsporozoite gene:amino acid sequence of repetitive epitope. Science 1984; 225:628-630.
30. Bernard N. Kanoi, Thomas G. Egwang. New concepts in vaccine development in malaria [J]. Current Opinion in Infectious Diseases 2007,20:311-316.
31. Gardner MJ,Hall N,Fung E,et al.Genome sequence of the human malaria parasite Plasmodium falciparum [J].Nature,2002;419:498-511.
32. Good M F,Berzofsky J A,Miller L H. The T cell response to the malaria circumsporozoite protein:an immunological approach to vaccine development [J].Ann Rev lmmunol,1988,6:663-688.
33. Michell G H. An update on candidate malaria vaccines [J]. Parasitol,1989,98 (S):29-47.
34. Dontfraid F,Cochran M A,Pombo D,et al.Human andmurine CD4 T cell epitopes map to the same region of the malaria circumsporozoite protein:limited immunogenicity of sporozoites and circumsporozoite protein [J].Mol Biol Med,1988,5 (3):185-196.
35.方建民,余新炳,徐劲.恶性疟原虫疫苗的研究Ⅰ-环子孢子蛋白基因中央保守区的体外扩增[J].寄生虫与医学昆虫学报,1994,1(3)：1-5.
36. Herrington D A,Losonsky GA,Smith G,et al. Safety and immunogenicity in volunteers of a recombinant Plasmodiumf alciparum circumsporozoite proteinmalaria vaccine produced in Lepidopteran cells[J].Vaccine,1992,10 (12):841-846.
37. Roggero MA, Weilenmann C, Bonelo A, et al.Plasmodium falciparum CSC terminal fragment: preclinical evaluation and phase I clinical studies. Parassitologia,1999,41(1-3):421-424.
38. Ballou WR,Cahill CP.Two decades of commitment to malaria vaccine development:glaxosmit hkline biologicals [J].The Ameri2can Society of Tropical Medicine and Hygiene,2007,77 (6):289-95.
39. Bojang KA, Milligan PJ, Pinder M, et al. Efficacy of RTS,S/AS02 malaria vaccine against Plasmodium falciparuminfection in semi-immune adult men in the Gambia:a randomised trial.Lancet,2001,358(9297):1927-1934.
40. Bojang KA. RTS,S/AS02A for malaria. Expert Rev Vaccines 2006; 5:611-615.
41. Grtchen V. A complex new vaccine shows promise [J]. Science,2004,306:587-9.
42. Gretchen V. A complex new vaccine shows promise. Science,2004,306(5696): 587-589.
43. Epstein JE, Charoenvit Y, Kester KE, et al. Safety, tolerability, and antibody responses in humans after sequential immunization with a PfCSP DNA vaccine followed by the recombinant proteinvaccine RTS,S/AS02A. Vaccine, 2004,22(13-14):1592-1603.
44. Jose AS,Gray D,Heppner J R. Phase 1 Safety and immunogenicity trial of malaria vaccine RTS,S/AS02A in adult s in a hyperendemic region of Western Kenya [J].Am J Trop Med Hyg,2006,75 (1):166-70.
45. Vasee SM, Egeruan Bl, Paul M, et al. A randomised, double-blind, controlled vaccine efficacy trial of DNA/MVA ME-TRAP against malaria infection in Gambian adults.Plos Med,2004,1(2):e33.
46.陈华,杨恒林.疟疾疫苗研究进展[J].中国病原生物学杂志,2010,5(3)：225-230.
47. Michael A, Ajit L, Sarah CG, et al. Cytotoxic T-lymphocyte epitopes for HLA-B53 and other HLA types in the malaria vaccine candidate liver-stage antigen 3.Infect Immun,2000,68(1)：227-232.
48.周家莲,杨恒林.抗疟药研究现状与发展趋势[J].中国病原生物学杂 志,2008,11(3)：865-7.
49.王华,谭光宏,黄风迎,等.伯氏疟原虫裂殖子表面蛋白PbMSP4/5基因高效植物表达载体的构建[J].现代预防医学,2009,36(7)：63-7.
50.赖秀球,朱家勇.恶性疟原虫裂殖子表面蛋白2融合HBS基因重组质粒的免疫原性研究[J].中国人兽共患病杂志.2003,19(3)：63-7.
51.高慧萍,张冬梅,潘卫庆.恶性疟原虫裂殖子表面蛋白3功能区片段的制备及其免疫原性分析[J].热带医学杂志.2008,12(8)：1199-202.
52.王瑞,钱锋,曲莉,等.恶性疟原虫融合抗原PfcP22.9的单克隆抗体制备及功能分析[J].中国寄生虫学与寄生虫病杂志,2006,24(4)：247-50.
53.张忠广,赵恒梅,宫玉秀.恶性疟原虫主要裂殖子表面蛋白1C未端片段在毕赤酵母表达系统的高效表达与纯化[J].中国人兽共患病杂志,2005,21(12)：1047-5
54. Yazdani SS, Shakri AR, Mukherjee P, et al. Evaluation of immune responses elicited in mice against a recombinant malaria vaccine based onPlasmodium vivax Duffy binding protein. Vaccine,2004,22(27-28):3727-3737.
55. Dontfrai D F, Cochran M A, Pomb O D, et al. Human and murine CD4 Tceil epi-topes map to the same region o f the malaria circum sporozoi e protein:lim-ited immunogenicity of sporozoite s and circum sporozoite protein [J]. Mol Biol Med,1988,5 (3):185-196.
56.黄加忠,李靖.疟疾候选疫苗研究进展情况综述[J].中国医药指南,2010,8(28)：40-42.
57. Theisen M,Dodoo D, Toure-Balde A,et al. Selection of glutamaterich protein long synthetic peptides for vaccine development:antigenicity and relationship with clinical protection and immunogenicity[J].Infect Immun,2001,69(9):5223-5229.
58. Schofield L. T cell immunity to malaria sporozoites[J]. Exp Parastol,1989,68 (3):357-364.
59. Good M F.A malaria vaccine strategy based on the induction of cellular immunity [J]. Immunolog y Today,1992,13(4):126-130.
60. Hajime H, Anthony WS, Takafumi T, et al. Antibodies to malaria vaccine candidates Pvs25 and Pvs28 completely block the ability of Plasmodium vivax to infect mosquitoes. Infect Immun,2000,68(12):6618-6623.
61. Jetsumon S, Takafumi T, Hajime H, et al. Blocking of transmission to mosquitoes by antibody toPlasmodium vivaxmalaria vaccine candidates Pvs25 and Pvs28 despite antigenic polymorphism in field isolates. Am J Trop Med Hyg, 2003,69(5):536-541.
62. Zou L, Miles AP, Wang J, et al. Expression of malaria transmission-blocking vaccine antigen Pfs25 inPichia pastorisfor use in human clinical trials. Vaccine, 2003,21(15):1650-1657.
63. Takeshi A,Ai K,Hitoshi O.Nasal immunization with a malaria transmission-blocking vaccine candidate,Pfs25,induces complete protective immunity in mice against field isolates ofPlasmodium falciparum[J].Infect lmmu,2005,11:7375-80.
64. Andre L,Petra S,Marcel D.Age-dependent distribution of Plasmodium falciparumgametocytes quantified by PFS25 real-time QT-Nasba in across-sectional Stydt in burkina faso[J].The American Society of Tropical Medicine and Hygiene,2007,76(4):626-30.
65. Godfree M,Jorge M,Nirbhay K.Murine model for assessment of Plasmodium falciparum transmission-blocking vaccine using transgenic Plasmodium bergheiparasites expressing the target antigen Pfs25[J]. Infect lmmu,2008,5:2018-24.
66. Fanning SL, Czesny B, Sedegah M, et al. A glycosylphosphatidylinositol anchor signal sequence enhances the immunogenicity of a DNA vaccine encodingPlasmodium falciparum sexual-stage antigen, Pfs230. Vaccine, 2003,21(23):3228-3235.
67. Eappen GA, Shabana I, Prakash S, et al. Plasmodium and Anopheles transcriptional repertoire during ookinete development and midgut invasion. J Biol Chem,2002,279(7):5573-5580.
68. Kashala O, Amador R, Valero MV, et al. Safety, tolerability and immunogenicity of new formulations of thePlasmodium falciparum malaria peptide vaccine SPf66 combined with the immunological adjuvant QS-21. Vaccine,2002,20 (17-18):2263-2277.
69. Patarroyo M E, Romoro P, Torres M L, et al. Using synthetic peptide experimental induction of protective immunity against Plasmodium falciparum. Nature, 1986,328:629-623.
70. Patarroyo M E, Amador R, Clavijo P, et al. A synthetic vaccine protects humans against challenge with axesual blood stage of Plasmodium falciparum malaria. Nature,1987,332:158-161.
71. Alonso PL, Smith T, Schellenberg JR, et al. Randomised trial of efficacy of SPf66 vaccine againstPlasmodium falciparummalaria in children in southern Tanzania. Lancet,1994,344(8931):1175-1181.
72.张冬梅,潘卫庆.重组恶性疟原虫红细胞结合抗原175功能区的制备及联合免疫.第二军医大学学报,2004,25(1)：14-17.
73. Meraldi V, Nebie I, Tiono AB, et al. Natural antibody response to Plasmodium falciparumExp-1, MSP-3 and GLURP long synthetic peptides and association with protection. Parasite lmmunol,2004,26(6-7):265-272.
74.马金友,余燕.疟疾疫苗的研究进展[J].安徽农业科学,2007,35(4)：951-952.
75. Zhou S, Liu S, Song G, et al.Protective immunity induced by the full-length cDNA encoding paramyosin of Chinese Schistosomajaponicum[J].Vaccine,2000,18 (27):3196-3204.
76. Liu SJ,Cheng JZ,Tang CW, et al.Construction and expression of DNA vaccine plRES-Sj97-Sj14-Sj26 and its immunogenicity in mice[J].J Huazhong Uni Sci Techn (Med sci),2007,27 (6):625-9.
77. Glyde DF.Immunity to falctparum and vivax malaria induced by trradiated review of the university of Maryland studies,1971-1975[M].Bull WHO, 1990.9-12.
78. Sedega HM, Hedstro MR, Hobar T P, et al. Protection against malaria by immu-nization with plasmid DNA encoding circum sporozoite protein[J].PNAS,1994, 91 (2):9866-9870.
79.钟辉,曹诚,李平,等.以恒河猴为模型的DNA疫苗的免疫保护作用研究.遗传学报,2000,27(2)：95-100.
80. Rainczuk A, Scorza T, Spithill TW, et al. A bicistronic DNA vaccine containing apical membrane antigen 1 and merozoite surface protein 4/5 can prime humoral and cellular immune responses and partially protect mice against virulentPlasmodium chabaudiadamiDS malaria. Infect Immun,2004,72(10): 5565-5573.
81. Kongkasuriyachai D, Bartels AL, Stowers A, et al. Potent immunogenicity of DNA vaccines encodingPlasmodium vivaxtransmission-blocking vaccine candidates Pvs25 and Pvs28-evaluation of homologous and heterologous antigen-delivery prime-boost strategy. Vaccine,2004,22(23-24):3205-3213.
82. Tine J A, Lanar D E, Smith D M, et al. NYV AC-Pf7:a poxvirus vectored, multian-tigen, multistage vaccine can didate for Plasmodium falciparum malaria[J]. Infe ctlmmun,1996,64 (9):3833-3844.
83. Eric P, Sarah CG, Joerg S, et al. APlasmodium falciparum candidate vaccine based on a six-antigen polyprotein encoded by recombinant poxviruses. Proc Natl Acad Sci USA,2004,101(1):290-295.
84.李学荣,余新炳,罗树红,等.恶性疟原虫重新质粒DNA接种诱导BALB/c小鼠的免疫应答[J].中国寄生虫学与寄生虫病杂志,1998,16(4)：241-245.
85.郑春福,吴少庭,陈雅棠,等.恶性疟原虫FCC-1/HN株裂殖子表面抗原2(MSA-2)基因在卡介苗BCG中的表达[J].寄生虫与医学昆虫学报,2002,9(4)：193-197.
86.钱锋,潘卫庆.在减毒伤寒杆菌CVD908疫苗株中四环素诱导表达恶性疟原虫MSP 1-31片段基因[J].第二军医大学学报,2002,23(1)：51-52.
87. Tang Y Y, Wang H. Construction and immunogenicity prediction of Plasmodium falciparum CTL epitope minigene vaccine[J].Science in China SerC,2001,44(2): 207-215.
88. Zhaoyang Y, Jenny H, Lisa R, et al. A retrogen strategy for pre-sentation of an intracellular tumor antigen as an exogenous antigen by dendritic cells induces potent antitumor T helper and CTL responses. Cancer Res, 2001,61(9):3704-3711.
89. Marian CB, Christl AD, Michael PA, et al. Cross-species interactions between malaria parasites in humans. Science,2000,287(5454):845-848.
90. Good MF, Stanisic D, XuH, et al.The immunological challenge to developing a vaccine to the blood stages of malaria parasites. Immunol Rev, 2004,201:254-267.
91. Urban BC, Ferguson DJ, Pain A, et al. Plasmodium falciparum infected erythrocytes modulate the maturation of dendritic cells. Nature, 1999,400:73-80.
92. Craig A, Scherf A. Molecules on the surface of the Plasmodium falciparum infected erythrocyte and their role in malaria pathogenesis and immune evasion. Mol Biochem Parasitol,2001,115:129-143.
93. Doolan DL, Aguiar JC, Weiss WR, et al. Utilization of genomic sequence information to develop malaria vaccines. J Exp Biol,2003,206:3789-3802.
94. Karine GL, Yingyao Z, Peter LB, et al. Discovery of gene function by expression profiling of the malaria parasite life cycle.Science,2003,301(5639):1503-1508
95. Ljungstron I, Perlmann H, et al. Methods in malaria research.2004, Manassas, Virginia.
96. Hoffman S, Oster C, PloweC, et al. Naturally acquired antibodies to sporozoites do not prevent malaria:vaccine development implications. Science 1987;237: 639-642.
97.Chougnet C, Troye-Blomberg M, Deloron P, et al. Human immune response in Plasmodium falciparum malaria. Synthetic peptides corresponding to known epitopes of the Pf155/RESA antigen induce production of parasite-specific antibodies in vitro. J Immunol1991; 147:2295-2301.
98. Di Perri G, Bonora S, Vento S, Concia E. Naturally acquired immunity to Plasmodium falciparum. Parasitol Today 1995; 11:346-347.
99. Bejon P, Kani OK, Mwacharo J, et al. Alternating vector immunizations encoding preerythrocytic malaria antigens enhance memory responses in a malaria endemic area. Eur J Immunol 2006; 36:2264-2272.
100. Liu CH, Fan YT, Dias A, et al. Cutting edge:dendritic cells are essential for invivo IL-12 production and development of resistance against Toxoplasma gondii infection in mice. J Immunol 2006; 177:31-35.
101. Pouniotis DS, Proudfoot O, Bogdanoska V, et al. Dendritic cells induce immunity and long-lasting protection against blood-stage malaria despite an in vitro parasite-induced maturation defect. Infect Immun 2004; 72:5331-5339.
102. Seixas E, Cross C, Quin S, Langhorne J. Direct activation of dendritic cells by the malaria parasite, Plasmodium chabaudi chabaudi. Eur J Immunol 2001;31:2970-2978.
103. Perlmann P, Troye-Blomberg M. Malaria blood-stage infection and its control by the immune system. Folia Biol (Praha) 2000; 46:210-218.
104. Bergmann ES, Ballou RW, Krzych U. Detection of CD4tCD45ROt T lymphocytes producing IL-4 in response to antigens on Plasmodium falciparum erythrocytes: an in vitro correlate of protective immunity induced with attenuated Plasmodium falciparum sporozoites. Cell Immunol 1997; 180:143-152.
105. Jangpatarapongsa K, Sirichaisinthop J, Sattabongkot J, et al. Memory T cells protect against Plasmodium vivax infection. Microbes Infect 2006; 8:680-686.
106. Schutyser E, Struyf S, Van Damme J. The CC chemokine CCL20 and its receptor CCR6. Cytokine Growth Factor Rev 2003; 14:409-426.
107. Chavan R, Marfatia KA, An IC, et al. Expression of CCL20 and granulocyte-macrophage colony-stimulating factor, but not Flt3-L, from modified vaccinia virus ankara enhances antiviral cellular and humoral immune responses. J Virol 2006; 80:7676-7687.
108. Robert Menard. Knockout malaria vaccine [J].2005;433:133-134.
109. Hoffman SL.Save the children [J].Nature,2004;430:940-941.
110. JM,Angiuoli SV,Suh BB,et al.Genome sequence and comparative analysis of the model rodent malaria parasite Plasmodium yoelii yoelii [J].Nature,2002, 419:512-519.
111. Menard. Targeting malaria vaccines? [J].Nature,2005;433:113-114.
112. Waters AP, Mota MM, van Dijk MR, et al. Malaria vaccines:back to the future [J]. Science,2005; 307:528-530.
113. Legler DE, Loetscher M, Roos RS, et al. B cell-attracting chemokine 1, a human CXC chemokine expressed in lymphoid tissues, selectively attracts B lymphocytes via BLR1/CCR5 [J]. J Exp Med,1998;87 (4):655-660.
114. Gunn MD,Ngo VN,Ansel KM,et al.B-cell-homing chemokine made in lymphoid follicles activates Burkitt's lymphoma receptor-1.Nature,1998;391 (6669):799.
115. Holmes WE,Lee J.,Kuang WJ,et al.Structure and functional expression of ahuman interleukin-8 receptor.Science,1991;253:1278-1280.
116. Wang JM, Deng XY, Gong WH, et al. Chemokines and their role in tumor growth and metastasis. J Immunol Methods,1998; 220:1-17.
117. Murphy PM,Baggiolini M,Charo IF,et al.Power international union of pharmacology. XXII. nomenclature for chemokine receptors. Pharmacol Rev,2000;52:145-176.
118. Appay V,Sarah L,Rowland-Jones.RANTES:a versatile and controversial chemokine. Trends in lmmunology,2001;22:83-87.
119. Hang FY, Li YN, Wang H, et al. A fusion protein containing murine vascular endothelial growth factor and tissue factor induces thrombogenesis and suppression of tumor growth in a colon carcinoma model [J]. J Zhejiang Univ Sci B;2008;9 (8):602-609.
120. Jiao JG, Li YN, Wang H, et al. A plasmid DNA vaccine encoding the extracellular domain of porcine endoglin induces anti-tumour immune response against self-endoglin-related angiogenesis in two liver cancer models [J]. Dig Liver Dis, 2006; 38 (8):578-587.
121.傅春华,田聆,魏于全,等.转染小鼠可溶性B淋巴细胞刺激因子稳定表达细胞株的筛选与鉴定.生物医学工程学杂志,2004,21(6)：897-900
122. Moser B and Loetscher P. Lymphocyte traffic control by chemokines [J]. Nat Immunol, Feb 2001; 2 (2):123-8.
123. Ansel KM, Ngo VN, Hyman PL, et al. A chemokine-driven positive feedback loop organizes lymphoid follicles [J]. Nature,2000; 406 (6793):309-314.
124. Dilloo D, Bacon K, Holden W, et al. Combined chemokine and cytokine gene transfer enhances antitumor immunity [J]. Nat Med,1996; 2 (10):1090-1095.
2. WHO World Malaria Report 2009[EB/OL].http://www.who.int/malaria/world_malaria_report_2009/en/index.html.
3. Kappe SH, Kaiser K, Matuschewski K. The Plasmodium sporozoite journey: a rite of passage [J]. Trends Parasitol,2003; 19:135-143.
4.李雍龙主编,人体寄生虫学(第七版)[M].北京：人民卫生出版社,2008.
5.周家莲,杨恒林.抗疟药研究现状与发展趋势[J].中国病原生物学杂志,2008,11(3)：865-7.
6. Marsh K, Kinyanjui S. Immune effector mechanisms in malaria [J]. Parasite Immunol,2006,28(1/2):51-60.
7.周华,孙立新,朱昌亮.疟疾疫苗研究的主要进展[J].国外医学寄生虫病分册,2005,32(2)：72-6.
8. Danie J, Carucci MC. Malaria vaccine research at the Naval Medical ResearchCenter.Navy Med,2001,5：23.
9.潘卫庆.疟疾疫苗研究的现状和展望[J].第二军医大学学报,2004,25(1)：1-4.
10. Nussenzweig RS,Vanderberg J,Most H,et al. Protective immunity produced by the injection of X-irradiated sporozoites of Plasmodium berghei [J]. Nature, 1967; 216:160-162.
11.朱艳琴,叶炳辉.疟疾疫苗的研究现状和难度[J].微生物学免疫学进展,1997,25(4)：74-6.
12. Thathy V., Menard R..Gene Targeting in Plasmodium berghei. Methods Mol Med,2002;72:317-331.
13. Girard MP, Reed ZH, FriedeM, KienyMP. A review of human vaccine research and development:malaria. Vaccine 2007; 25:1567-1580.
14. Renia L, Gruner AC, Mauduit M, Snounou G. Vaccination against malaria with live parasites. Expert Rev Vaccines 2006; 5:473-481.
15. SedegahM, Hoffman SL. Immunological responses of neonates and infants to DNA vaccines. Methods Mol Med 2006; 127:239-251.
16. Matuschewski K. Vaccine development against malaria. Curr Opin Immunol 2006; 18:449-457.
17. Holling Dale M R. Antisporozoite antibodies[J]. Bull WHO,1990,68 (5):47-48.
18. Hoffman SL,Goh LM,Luke TC,et al.Protection of humans against malaria by immunization with radiation-attenuated Plasmodium falciparum sporozoites [J]. J Infect Dis,2002;185:1155-64.
19. Glyde D F. Immunity to Plasmodium falciparum an vivax malaria induced by irra-diated sporozoites:are view of the university o f Maryland studies[J]. Bull WHO,1990,68 (5):9-12.
20. Srivastava K, Singh S, Singh P, Puri SK. In vitro cultivation of Plasmodium falciparum:studies with modified medium supplemented with ALBUMAX II and various animal sera. Exp Parasitol 2006; [Epub ahead of print].
21. Balu B, Adams JH. Advancements in transfection technologies for plasmodium. Int J Parasitol 2007; 37:1-10.
22. Scheller LF,Azad AF.Maintenance of protective immunity against malaria by persistent hepatic parasites derived from irradiated sporozoites [J]. Proc Natl Acad Sci USA,1995;92:4066-4068.
23. Luke TC, Hoffman SL. Rationale and plans for developing a nonreplicating, metabolically active, radiation-attenuated Plasmodium falciparum sporozoite vaccine. J Exp Biol 2003; 206:3803-3808.
24. Mueller AK, Labaied M, Kappe SH, et al. Genetically modified Plasmodium parasites as a protective experimental malaria vaccine [J]. Nature,2005; 433 (7022):164-167.
25. Mueller AK,Camargo N,Kaiser K,et al.Plasmodium liver stage developmental arrest by depletion of a protein at the parasite-host interface[J].Proc Natl Acad Sci USA,2005,102(8):3022-3027.
26. Van Dijk MR, Douradinha B, Franke-Fayard B,et al. Genetically attenuated, P36p-deficient malarial sporozoites induce protective immunity and apoptosis of infected liver cells[J].Proc Natl Acad Sci USA,2005,102(34):12194-12199.
27. Daubersies P,Thomas AW,Millet P, et al.Protection against Plasmodium falciparum malaria in chimpanzees by immunization with the conserved pre-erythrocytic liver-stage antigen 3 [J]. Nat Med,2000;6:1258-63.
28. Alonso PL,Sacarlal J,Aponte JJ,et al.Efficacy of the RTS,S/AS02A vaccine against Plasmodium falciparum infection and disease in young African children: randomized controlled trial [J]. Lancet,2004;364:1411-1420.
29. Enea V, Ellis J, Zavala F, et al. DNA cloning of Plasmodium falciparum circumsporozoite gene:amino acid sequence of repetitive epitope. Science 1984; 225:628-630.
30. Bernard N. Kanoi, Thomas G. Egwang. New concepts in vaccine development in malaria [J]. Current Opinion in Infectious Diseases 2007,20:311-316.
31. Gardner MJ,Hall N,Fung E,et al.Genome sequence of the human malaria parasite Plasmodium falciparum [J].Nature,2002;419:498-511.
32. Good M F,Berzofsky J A,Miller L H. The T cell response to the malaria circumsporozoite protein:an immunological approach to vaccine development [J].Ann Rev lmmunol,1988,6:663-688.
33. Michell G H. An update on candidate malaria vaccines [J]. Parasitol,1989,98 (S):29-47.
34. Dontfraid F,Cochran M A,Pombo D,et al.Human andmurine CD4 T cell epitopes map to the same region of the malaria circumsporozoite protein:limited immunogenicity of sporozoites and circumsporozoite protein [J].Mol Biol Med,1988,5 (3):185-196.
35.方建民,余新炳,徐劲.恶性疟原虫疫苗的研究Ⅰ-环子孢子蛋白基因中央保守区的体外扩增[J].寄生虫与医学昆虫学报,1994,1(3)：1-5.
36. Herrington D A,Losonsky GA,Smith G,et al. Safety and immunogenicity in volunteers of a recombinant Plasmodiumf alciparum circumsporozoite proteinmalaria vaccine produced in Lepidopteran cells[J].Vaccine,1992,10 (12):841-846.
37. Roggero MA, Weilenmann C, Bonelo A, et al.Plasmodium falciparum CSC terminal fragment: preclinical evaluation and phase I clinical studies. Parassitologia,1999,41(1-3):421-424.
38. Ballou WR,Cahill CP.Two decades of commitment to malaria vaccine development:glaxosmit hkline biologicals [J].The Ameri2can Society of Tropical Medicine and Hygiene,2007,77 (6):289-95.
39. Bojang KA, Milligan PJ, Pinder M, et al. Efficacy of RTS,S/AS02 malaria vaccine against Plasmodium falciparuminfection in semi-immune adult men in the Gambia:a randomised trial.Lancet,2001,358(9297):1927-1934.
40. Bojang KA. RTS,S/AS02A for malaria. Expert Rev Vaccines 2006; 5:611-615.
41. Grtchen V. A complex new vaccine shows promise [J]. Science,2004,306:587-9.
42. Gretchen V. A complex new vaccine shows promise. Science,2004,306(5696): 587-589.
43. Epstein JE, Charoenvit Y, Kester KE, et al. Safety, tolerability, and antibody responses in humans after sequential immunization with a PfCSP DNA vaccine followed by the recombinant proteinvaccine RTS,S/AS02A. Vaccine, 2004,22(13-14):1592-1603.
44. Jose AS,Gray D,Heppner J R. Phase 1 Safety and immunogenicity trial of malaria vaccine RTS,S/AS02A in adult s in a hyperendemic region of Western Kenya [J].Am J Trop Med Hyg,2006,75 (1):166-70.
45. Vasee SM, Egeruan Bl, Paul M, et al. A randomised, double-blind, controlled vaccine efficacy trial of DNA/MVA ME-TRAP against malaria infection in Gambian adults.Plos Med,2004,1(2):e33.
46.陈华,杨恒林.疟疾疫苗研究进展[J].中国病原生物学杂志,2010,5(3)：225-230.
47. Michael A, Ajit L, Sarah CG, et al. Cytotoxic T-lymphocyte epitopes for HLA-B53 and other HLA types in the malaria vaccine candidate liver-stage antigen 3.Infect Immun,2000,68(1)：227-232.
48.周家莲,杨恒林.抗疟药研究现状与发展趋势[J].中国病原生物学杂 志,2008,11(3)：865-7.
49.王华,谭光宏,黄风迎,等.伯氏疟原虫裂殖子表面蛋白PbMSP4/5基因高效植物表达载体的构建[J].现代预防医学,2009,36(7)：63-7.
50.赖秀球,朱家勇.恶性疟原虫裂殖子表面蛋白2融合HBS基因重组质粒的免疫原性研究[J].中国人兽共患病杂志.2003,19(3)：63-7.
51.高慧萍,张冬梅,潘卫庆.恶性疟原虫裂殖子表面蛋白3功能区片段的制备及其免疫原性分析[J].热带医学杂志.2008,12(8)：1199-202.
52.王瑞,钱锋,曲莉,等.恶性疟原虫融合抗原PfcP22.9的单克隆抗体制备及功能分析[J].中国寄生虫学与寄生虫病杂志,2006,24(4)：247-50.
53.张忠广,赵恒梅,宫玉秀.恶性疟原虫主要裂殖子表面蛋白1C未端片段在毕赤酵母表达系统的高效表达与纯化[J].中国人兽共患病杂志,2005,21(12)：1047-5
54. Yazdani SS, Shakri AR, Mukherjee P, et al. Evaluation of immune responses elicited in mice against a recombinant malaria vaccine based onPlasmodium vivax Duffy binding protein. Vaccine,2004,22(27-28):3727-3737.
55. Dontfrai D F, Cochran M A, Pomb O D, et al. Human and murine CD4 Tceil epi-topes map to the same region o f the malaria circum sporozoi e protein:lim-ited immunogenicity of sporozoite s and circum sporozoite protein [J]. Mol Biol Med,1988,5 (3):185-196.
56.黄加忠,李靖.疟疾候选疫苗研究进展情况综述[J].中国医药指南,2010,8(28)：40-42.
57. Theisen M,Dodoo D, Toure-Balde A,et al. Selection of glutamaterich protein long synthetic peptides for vaccine development:antigenicity and relationship with clinical protection and immunogenicity[J].Infect Immun,2001,69(9):5223-5229.
58. Schofield L. T cell immunity to malaria sporozoites[J]. Exp Parastol,1989,68 (3):357-364.
59. Good M F.A malaria vaccine strategy based on the induction of cellular immunity [J]. Immunolog y Today,1992,13(4):126-130.
60. Hajime H, Anthony WS, Takafumi T, et al. Antibodies to malaria vaccine candidates Pvs25 and Pvs28 completely block the ability of Plasmodium vivax to infect mosquitoes. Infect Immun,2000,68(12):6618-6623.
61. Jetsumon S, Takafumi T, Hajime H, et al. Blocking of transmission to mosquitoes by antibody toPlasmodium vivaxmalaria vaccine candidates Pvs25 and Pvs28 despite antigenic polymorphism in field isolates. Am J Trop Med Hyg, 2003,69(5):536-541.
62. Zou L, Miles AP, Wang J, et al. Expression of malaria transmission-blocking vaccine antigen Pfs25 inPichia pastorisfor use in human clinical trials. Vaccine, 2003,21(15):1650-1657.
63. Takeshi A,Ai K,Hitoshi O.Nasal immunization with a malaria transmission-blocking vaccine candidate,Pfs25,induces complete protective immunity in mice against field isolates ofPlasmodium falciparum[J].Infect lmmu,2005,11:7375-80.
64. Andre L,Petra S,Marcel D.Age-dependent distribution of Plasmodium falciparumgametocytes quantified by PFS25 real-time QT-Nasba in across-sectional Stydt in burkina faso[J].The American Society of Tropical Medicine and Hygiene,2007,76(4):626-30.
65. Godfree M,Jorge M,Nirbhay K.Murine model for assessment of Plasmodium falciparum transmission-blocking vaccine using transgenic Plasmodium bergheiparasites expressing the target antigen Pfs25[J]. Infect lmmu,2008,5:2018-24.
66. Fanning SL, Czesny B, Sedegah M, et al. A glycosylphosphatidylinositol anchor signal sequence enhances the immunogenicity of a DNA vaccine encodingPlasmodium falciparum sexual-stage antigen, Pfs230. Vaccine, 2003,21(23):3228-3235.
67. Eappen GA, Shabana I, Prakash S, et al. Plasmodium and Anopheles transcriptional repertoire during ookinete development and midgut invasion. J Biol Chem,2002,279(7):5573-5580.
68. Kashala O, Amador R, Valero MV, et al. Safety, tolerability and immunogenicity of new formulations of thePlasmodium falciparum malaria peptide vaccine SPf66 combined with the immunological adjuvant QS-21. Vaccine,2002,20 (17-18):2263-2277.
69. Patarroyo M E, Romoro P, Torres M L, et al. Using synthetic peptide experimental induction of protective immunity against Plasmodium falciparum. Nature, 1986,328:629-623.
70. Patarroyo M E, Amador R, Clavijo P, et al. A synthetic vaccine protects humans against challenge with axesual blood stage of Plasmodium falciparum malaria. Nature,1987,332:158-161.
71. Alonso PL, Smith T, Schellenberg JR, et al. Randomised trial of efficacy of SPf66 vaccine againstPlasmodium falciparummalaria in children in southern Tanzania. Lancet,1994,344(8931):1175-1181.
72.张冬梅,潘卫庆.重组恶性疟原虫红细胞结合抗原175功能区的制备及联合免疫.第二军医大学学报,2004,25(1)：14-17.
73. Meraldi V, Nebie I, Tiono AB, et al. Natural antibody response to Plasmodium falciparumExp-1, MSP-3 and GLURP long synthetic peptides and association with protection. Parasite lmmunol,2004,26(6-7):265-272.
74.马金友,余燕.疟疾疫苗的研究进展[J].安徽农业科学,2007,35(4)：951-952.
75. Zhou S, Liu S, Song G, et al.Protective immunity induced by the full-length cDNA encoding paramyosin of Chinese Schistosomajaponicum[J].Vaccine,2000,18 (27):3196-3204.
76. Liu SJ,Cheng JZ,Tang CW, et al.Construction and expression of DNA vaccine plRES-Sj97-Sj14-Sj26 and its immunogenicity in mice[J].J Huazhong Uni Sci Techn (Med sci),2007,27 (6):625-9.
77. Glyde DF.Immunity to falctparum and vivax malaria induced by trradiated review of the university of Maryland studies,1971-1975[M].Bull WHO, 1990.9-12.
78. Sedega HM, Hedstro MR, Hobar T P, et al. Protection against malaria by immu-nization with plasmid DNA encoding circum sporozoite protein[J].PNAS,1994, 91 (2):9866-9870.
79.钟辉,曹诚,李平,等.以恒河猴为模型的DNA疫苗的免疫保护作用研究.遗传学报,2000,27(2)：95-100.
80. Rainczuk A, Scorza T, Spithill TW, et al. A bicistronic DNA vaccine containing apical membrane antigen 1 and merozoite surface protein 4/5 can prime humoral and cellular immune responses and partially protect mice against virulentPlasmodium chabaudiadamiDS malaria. Infect Immun,2004,72(10): 5565-5573.
81. Kongkasuriyachai D, Bartels AL, Stowers A, et al. Potent immunogenicity of DNA vaccines encodingPlasmodium vivaxtransmission-blocking vaccine candidates Pvs25 and Pvs28-evaluation of homologous and heterologous antigen-delivery prime-boost strategy. Vaccine,2004,22(23-24):3205-3213.
82. Tine J A, Lanar D E, Smith D M, et al. NYV AC-Pf7:a poxvirus vectored, multian-tigen, multistage vaccine can didate for Plasmodium falciparum malaria[J]. Infe ctlmmun,1996,64 (9):3833-3844.
83. Eric P, Sarah CG, Joerg S, et al. APlasmodium falciparum candidate vaccine based on a six-antigen polyprotein encoded by recombinant poxviruses. Proc Natl Acad Sci USA,2004,101(1):290-295.
84.李学荣,余新炳,罗树红,等.恶性疟原虫重新质粒DNA接种诱导BALB/c小鼠的免疫应答[J].中国寄生虫学与寄生虫病杂志,1998,16(4)：241-245.
85.郑春福,吴少庭,陈雅棠,等.恶性疟原虫FCC-1/HN株裂殖子表面抗原2(MSA-2)基因在卡介苗BCG中的表达[J].寄生虫与医学昆虫学报,2002,9(4)：193-197.
86.钱锋,潘卫庆.在减毒伤寒杆菌CVD908疫苗株中四环素诱导表达恶性疟原虫MSP 1-31片段基因[J].第二军医大学学报,2002,23(1)：51-52.
87. Tang Y Y, Wang H. Construction and immunogenicity prediction of Plasmodium falciparum CTL epitope minigene vaccine[J].Science in China SerC,2001,44(2): 207-215.
88. Zhaoyang Y, Jenny H, Lisa R, et al. A retrogen strategy for pre-sentation of an intracellular tumor antigen as an exogenous antigen by dendritic cells induces potent antitumor T helper and CTL responses. Cancer Res, 2001,61(9):3704-3711.
89. Marian CB, Christl AD, Michael PA, et al. Cross-species interactions between malaria parasites in humans. Science,2000,287(5454):845-848.
90. Good MF, Stanisic D, XuH, et al.The immunological challenge to developing a vaccine to the blood stages of malaria parasites. Immunol Rev, 2004,201:254-267.
91. Urban BC, Ferguson DJ, Pain A, et al. Plasmodium falciparum infected erythrocytes modulate the maturation of dendritic cells. Nature, 1999,400:73-80.
92. Craig A, Scherf A. Molecules on the surface of the Plasmodium falciparum infected erythrocyte and their role in malaria pathogenesis and immune evasion. Mol Biochem Parasitol,2001,115:129-143.
93. Doolan DL, Aguiar JC, Weiss WR, et al. Utilization of genomic sequence information to develop malaria vaccines. J Exp Biol,2003,206:3789-3802.
94. Karine GL, Yingyao Z, Peter LB, et al. Discovery of gene function by expression profiling of the malaria parasite life cycle.Science,2003,301(5639):1503-1508
95. Ljungstron I, Perlmann H, et al. Methods in malaria research.2004, Manassas, Virginia.
96. Hoffman S, Oster C, PloweC, et al. Naturally acquired antibodies to sporozoites do not prevent malaria:vaccine development implications. Science 1987;237: 639-642.
97.Chougnet C, Troye-Blomberg M, Deloron P, et al. Human immune response in Plasmodium falciparum malaria. Synthetic peptides corresponding to known epitopes of the Pf155/RESA antigen induce production of parasite-specific antibodies in vitro. J Immunol1991; 147:2295-2301.
98. Di Perri G, Bonora S, Vento S, Concia E. Naturally acquired immunity to Plasmodium falciparum. Parasitol Today 1995; 11:346-347.
99. Bejon P, Kani OK, Mwacharo J, et al. Alternating vector immunizations encoding preerythrocytic malaria antigens enhance memory responses in a malaria endemic area. Eur J Immunol 2006; 36:2264-2272.
100. Liu CH, Fan YT, Dias A, et al. Cutting edge:dendritic cells are essential for invivo IL-12 production and development of resistance against Toxoplasma gondii infection in mice. J Immunol 2006; 177:31-35.
101. Pouniotis DS, Proudfoot O, Bogdanoska V, et al. Dendritic cells induce immunity and long-lasting protection against blood-stage malaria despite an in vitro parasite-induced maturation defect. Infect Immun 2004; 72:5331-5339.
102. Seixas E, Cross C, Quin S, Langhorne J. Direct activation of dendritic cells by the malaria parasite, Plasmodium chabaudi chabaudi. Eur J Immunol 2001;31:2970-2978.
103. Perlmann P, Troye-Blomberg M. Malaria blood-stage infection and its control by the immune system. Folia Biol (Praha) 2000; 46:210-218.
104. Bergmann ES, Ballou RW, Krzych U. Detection of CD4tCD45ROt T lymphocytes producing IL-4 in response to antigens on Plasmodium falciparum erythrocytes: an in vitro correlate of protective immunity induced with attenuated Plasmodium falciparum sporozoites. Cell Immunol 1997; 180:143-152.
105. Jangpatarapongsa K, Sirichaisinthop J, Sattabongkot J, et al. Memory T cells protect against Plasmodium vivax infection. Microbes Infect 2006; 8:680-686.
106. Schutyser E, Struyf S, Van Damme J. The CC chemokine CCL20 and its receptor CCR6. Cytokine Growth Factor Rev 2003; 14:409-426.
107. Chavan R, Marfatia KA, An IC, et al. Expression of CCL20 and granulocyte-macrophage colony-stimulating factor, but not Flt3-L, from modified vaccinia virus ankara enhances antiviral cellular and humoral immune responses. J Virol 2006; 80:7676-7687.
108. Robert Menard. Knockout malaria vaccine [J].2005;433:133-134.
109. Hoffman SL.Save the children [J].Nature,2004;430:940-941.
110. JM,Angiuoli SV,Suh BB,et al.Genome sequence and comparative analysis of the model rodent malaria parasite Plasmodium yoelii yoelii [J].Nature,2002, 419:512-519.
111. Menard. Targeting malaria vaccines? [J].Nature,2005;433:113-114.
112. Waters AP, Mota MM, van Dijk MR, et al. Malaria vaccines:back to the future [J]. Science,2005; 307:528-530.
113. Legler DE, Loetscher M, Roos RS, et al. B cell-attracting chemokine 1, a human CXC chemokine expressed in lymphoid tissues, selectively attracts B lymphocytes via BLR1/CCR5 [J]. J Exp Med,1998;87 (4):655-660.
114. Gunn MD,Ngo VN,Ansel KM,et al.B-cell-homing chemokine made in lymphoid follicles activates Burkitt's lymphoma receptor-1.Nature,1998;391 (6669):799.
115. Holmes WE,Lee J.,Kuang WJ,et al.Structure and functional expression of ahuman interleukin-8 receptor.Science,1991;253:1278-1280.
116. Wang JM, Deng XY, Gong WH, et al. Chemokines and their role in tumor growth and metastasis. J Immunol Methods,1998; 220:1-17.
117. Murphy PM,Baggiolini M,Charo IF,et al.Power international union of pharmacology. XXII. nomenclature for chemokine receptors. Pharmacol Rev,2000;52:145-176.
118. Appay V,Sarah L,Rowland-Jones.RANTES:a versatile and controversial chemokine. Trends in lmmunology,2001;22:83-87.
119. Hang FY, Li YN, Wang H, et al. A fusion protein containing murine vascular endothelial growth factor and tissue factor induces thrombogenesis and suppression of tumor growth in a colon carcinoma model [J]. J Zhejiang Univ Sci B;2008;9 (8):602-609.
120. Jiao JG, Li YN, Wang H, et al. A plasmid DNA vaccine encoding the extracellular domain of porcine endoglin induces anti-tumour immune response against self-endoglin-related angiogenesis in two liver cancer models [J]. Dig Liver Dis, 2006; 38 (8):578-587.
121.傅春华,田聆,魏于全,等.转染小鼠可溶性B淋巴细胞刺激因子稳定表达细胞株的筛选与鉴定.生物医学工程学杂志,2004,21(6)：897-900
122. Moser B and Loetscher P. Lymphocyte traffic control by chemokines [J]. Nat Immunol, Feb 2001; 2 (2):123-8.
123. Ansel KM, Ngo VN, Hyman PL, et al. A chemokine-driven positive feedback loop organizes lymphoid follicles [J]. Nature,2000; 406 (6793):309-314.
124. Dilloo D, Bacon K, Holden W, et al. Combined chemokine and cytokine gene transfer enhances antitumor immunity [J]. Nat Med,1996; 2 (10):1090-1095.